Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 50-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122 •) cofactor which, in the presence of S and E, oxidizes C 439 in α to a thiyl radical, C439•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y122• → W48 → Y356 in β2 to Y731 → Y730 → C439 in α2. 3-Aminotyrosine (NH2Y) has been sitespecifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y•) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y 356F-β2:Y731NH2Y-α2, Y 356F-β2:Y730NH2Y-α2, and wt-β2:Y731F/Y730NH2Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y• was detected. To study off-pathway oxidation, Y413, located 5Å from Y 730 and Y731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413• was sought in three complexes: wt-β2:Y413NH2Y-α2 (a), wt-β2:Y731F/Y413NH2Y-α2 (b), and Y356F-β2: Y413NH2Y-α2 (c). With (a), NH2Y• was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant forNH2Y• formation was 0.2-0.3% of that observed at 731 and 730, and with (c), noNH 2Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C439.
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